Novel carbazolyl–thiazolyl–chromone and carbazolyl–thiazolyl–pyrazole hybrids: synthesis, cytotoxicity evaluation and molecular docking studies

A simple synthetic method was performed to design a novel series of polycyclic systems consisting of carbazole–thiazolidinone–chromone hybrids 4a–e and carbazole–thiazolidinone–pyrazole hybrids 5a–e in excellent yields. The methodology depended on the one-pot four-component reaction of 3-amino-9-ethylcarbazole, substituted isothiocyanates, ethyl bromoacetate and 6-methyl-3-formylchromone in ethanol under ultrasound waves at 50 °C to give the carbazole–thiazolidinone–chromone hybrids 4a–e. The latter isolated products were treated with hydrazine hydrate in ethanol under ultrasound waves at 50 °C affording the corresponding carbazole–thiazolidinone–pyrazole hybrids 5a–e. Spectral and analytical data confirmed the structures of all the synthesized compounds. The target compounds were screened for their in vitro anticancer activities against HCT116, PC3 and HepG2 cancer cell lines using the standard SRB method. Fortunately, both compounds 5dand5e were the most active against all cancer cell lines compared with doxorubicin and can be promising anticancer agents. Both bioactive products 5band5e were studied by the molecular docking to see how they bind with VEGFR-2 receptor. The results indicated that those compounds exhibited high affinities towards VEGFR-2 and established remarkably similar interactions to those of the powerful VEGFR-2-KDR.


Introduction
Cancer disease is the leading cause of morbidity and mortality in the world.Cancer is inuenced by a number of factors such as genes, sex, age, food habits, stress, radiation and chemical agents. 1,2Recent advances in our understanding of cancer's molecular pathways have fueled a surge in cancer drug design research.This has led to the evaluation of diverse small molecule templates as potential cancer therapies. 3,4With these advances and ndings, a number of pharmaceuticals are now oen used to treat cancer.Although these medications have demonstrably extended patient lifespans, treatment success is not at the expected level.In recent years, researchers worked on specically tumor-targeted substances to achieve the required levels.In the development of cancer drugs, design of a hybrid molecule has gained importance. 5 The molecular hybridization methodology depends on the merging of pharmacophoric groups of multiple bioactive compounds in one molecular frame to create a unique hybrid product that is more active and less toxic than currently available pharmaceuticals.Hybrid molecules possess multiple implications in chemistry, pharmacology, and medicine.[8][9][10] Carbazoles are one of the most widespread substances in structures of bioactive compounds.2][13][14][15][16][17] Drug researchers have demonstrated an abundance of interest in carbazole-based hybrid compounds because they have a considerable inhibitory effect on a number of cancer cell lines when possessing heterocyclic systems especially thiazole and pyrazole at the C-3 position (Fig. 1).The anticancer properties of carbazole moiety are well recognized, and its associations with other moieties can improve its bioactive properties, while one core may exhibit anticancer effects, and the other cores may inhibit the cross-talk the way. 18][26][27] In contrast to traditional methods, multicomponent reactions (MCRs) offer a powerful and efficient approach in organic synthesis.][40][41][42][43] In the present study, we synthesized novel molecular frames by combining carbazole and thiazolidinone cores with either chromone or pyrazole core.The synthesis of the target compounds was designed in one-pot four-component reaction with the help of ultrasound waves.These compounds were then evaluated for their ability to tumor HCT116, PC3 and HepG2 cancer cell lines.
When the four-components reacted under catalyst-and solvent-free conditions for 3 h at room temperature or 75 °C, no product was isolated (Table 1, entries 1 and 2).When the reaction was performed without a catalyst in the presence of benzene or methanol or absolute ethanol at 50 °C, it gave the desired product 4a in 12%, 18% and 22% yields, respectively (Table 1, entries 3-5), while in the presence of absolute ethanol at 75 °C for 3 h, the product 4a was obtained in 41% yield (Table 1, entry 6).Thus, we decided to use some bases such as DBU, pyridine and Et 3 N (1.0 mmol) (Table 1, entries 7-9).It was found that Et 3 N as a catalyst led to the considerable product 4a in 52% yield aer 3 h (Table 1, entry 9).Therefore, the effect of the quantity of Et 3 N was examined.Increasing of the amount of the catalyst into 2.0 mmol increased the yield of the product to 75%  (Table 1, entry 10).Next, we tried to do this reaction in a shorter time with better yield by using ultrasonication effect.When the target reaction was carried out by four-components reaction in the presence of Et 3 N (2.0 mmol) under ultrasound irradiation at room temperature, it gave the product 4a in 80% yield aer 25 minutes (Table 1, entry 11).But, when the reaction was repeated under the same reaction condition at 50 °C, it gave the nal product 4a in 91% as excellent yield aer 50 min (Table 1, entry 12).The yield did not progress above 91% although increasing the time into 75 minutes and temperature into 75 °C (Table 1, entry 13).

Anticancer properties
Evaluation of cytotoxicity properties.We evaluated the effect of new compounds (4a-e and 5a-e) and the standard drug (doxorubicin) on the growth of various tumor cancer cells.We used the sulforhodamine B (SRB) assay to measure this effect on three different cancer cell lines (HCT116, PC3, and HepG2) and a normal healthy cell line from a mouse (3T3-L1). 51,52The IC 50 values for all synthesized compounds and doxorubicin are shown in Table 2.As per National Cancer Institute guidelines, a IC 50 value of #10 mg ml −1 was considered to demonstrate highly signicant inhibitory activity.Among the tested compounds on HCT-116 cancer cells, compound 5b emerged as the most potent inhibitor of cell growth with the lowest IC 50 value at 28.5 ± 1.5 mg ml −1 .Conversely, compound 5a displayed the weakest effect by its highest IC 50 value at 69.5 ± 1.5 mg ml −1 .The other compounds, 5c and 5e exhibited moderate antiproliferative activity with IC 50 values at 30.9 ± 2.3 and 39.9 ± 0.1 mg ml −1 , respectively.As for PC3 cells, both compounds 5b and 5e showed the highest antiproliferative activities with the smallest IC 50 values at 4.9 ± 0.8 and 9.2 ± 0.4 mg ml −1 , respectively, while compounds 5a and 5c displayed acceptable activities with IC 50 value at 12.9 ± 3.8 and 37.4 ± 1.6 mg ml −1 , respectively.Furthermore, compound 5e seems to be quite versatile.It demonstrated the most potent activity against HepG-2 cells with an impressive IC 50 value at 9.1 ± 0.5 mg ml −1 .
In addition, compounds 5a-c showed good activities against HepG-2 cells with IC 50 values between 18.5 ± 0.4 and 25.3 ± 1.4 mg ml −1 .The selectivity of these promising synthesized compounds (5a, 5b, 5c and 5e) in thier cytotoxicity between cancer and healthy cells is very important to be a chemotherapeutic agent.Here, their antiproliferative effects on the normal mouse broblast cell line, 3T3-L1 were evaluated.The order of toxicity power of these compounds on 3T3-L1 cells was 5c > 5a > 5e > 5b.This suggested that both compound 5b and 5e may be effective against a broader range of cancer cell lines.
Structure-activity relationship (SAR).The study also explored the relationship between the chemical structures of the compounds and their anticancer activity against the tested cell lines (HCT116, PC3, and HepG2).They found that the presence of a pyrazole ring compared to a chromone ring connected with the carbazole-thiazolidinone scaffold led to a more effective pharmacophore.In simpler terms, the specic types of cancer lines.Interestingly, considering the contribution of the group attached to the thiazolidinone ring at position 3 affected on the anticancer activity against all cancer cell lines.For HCT116 and PC3 cancer cells, allyl group was found to be the most effective than the other groups.In contrast to PC3 and HepG2 cancer cells, the 4-chlorophenyl group caused signicantly increased in the anticancer activity in comparison with the other groups.Further, the allyl and 4-chlorophenyl groups caused low cytotoxicity on the normal cell 3T3-L1 cells.This indicated great importance to understand both the type of cell and the structure of the compound that could be targeted by anticancer agent.Molecular docking study.A molecular docking approach has become a crucial strategy that provides the most promising route for drug designing and discovery. 53It is used to predict the strength of binding affinity between a drug target and ligand molecule.Additionally, this computational tool is more economic, time saving and effective over conventional technologies.According to the cytotoxicity results, this study was applied for the most effective compounds 5b and 5e to explore their binding mode towards vascular endothelial growth factor Kinase Insert Domain Receptor.The molecular docking results suggested that the synthesized compounds bind to VEGFR-2 in a similar way to a known VEGFR-2 inhibitor called K11 as shown in Table 3. [54][55][56][57][58][59] Fig. 4 illustrates how K11 interacted with VEGFR-2.It formed two hydrogen bonds with key amino acids CYS 919 and GLU 885, and its uorine atoms interacted with another key amino acid ILE 1044.In addition, it formed several Piinteractions including Pi-sulfur (CYS 1045), two Pi-sigma (VAL 848, LEU 1035), Pi-cation (LYS 868), and Pi-Pi T shaped (PHE 1047).According to the molecular docking results, both compounds 5b and 5e interacted with the VEGFR-2 protein in very similar binding modes, while their binding are −11.2 and −10.1 kcal mol −1 , respectively.Both 5b and 5e tted into the same pocket on the VEGFR-2 protein.Compound 5b revealed several modes of interactions including hydrogen bonds with GLU 917, THR 916 through NH group of pyrazole ring and the C]O group of the thiazole ring, respectively.Moreover, some Pi-interactions were observed including Pi-cation with LYS 868 through the thiazole ring, Pi-anion with ASP 1046 through the phenyl ring of carbazole moiety and Pi-alkyl with carbazole or thiazole moieties through the amino acids VAL 848 and LEU 889.Also, Pi-Pi T shaped interaction was observed with residual key PHE 1047 through the thiazole ring.With respect to product 5e, it displayed seven modes of interactions involving hydrogen bond with the amino acid THR 916 through C]O group of thiazole ring.Besides, it formed Pi-sigma interactions through carbazole, thiazole and pyrazole moieties with amino acids LEU 1035 and VAL 848.In addition, it formed Pi-cation, Pi-sulfur and Pi-alkyl interactions with key residue LYS 868, CYS 919, CYS 1045 and LEU 889, respectively through 4-chlorophenyl ring.From the above mentioned, the bioactive synthesized compounds 5b and 5e showed high affinities towards VEGFR-2 which correlated well with the results obtained from experimental cytotoxicity.Therefore, these compounds can be considered promising anticancer agents Fig. 5 and 6

General marks
The melting points were determined in an open capillary tube on a digital Stuart SMP-3 apparatus.IR spectra were measured on FT-IR (Nicolet IS10) spectrophotometer using ATR technique.The 1 H-and 13 C-NMR spectra were measured a Bruker spectrometer (400 and 100 MHz), using DMSO-d 6 as a solvent and TMS (d) as an internal standard.Mass spectra were recorded on direct probe controller inlet part to single quadropole mass analyzer in (Thermo Scientic GCMS).Elemental microanalyses were performed PerkinElmer 2400II at the Chemical War department, Ministry of Defense.The purity of the synthesized compounds was checked by thin layer chromatography (TLC) and elemental microanalysis.All the used ne chemicals and solvents were purchased form Sigma-Aldrich company and used without further purication.
A mixture of 3-amino-9-ethylcarbazole (1) (2.5 mmol, 0.525 g) and alkyl/aryl isothiocyanates 2a-e (2.5 mmol) was dissolved in absolute ethanol (20 ml) and irradiated under ultrasonication waves for 15 min at 50 °C.Then, adding a solution of ethyl bromoacetate (2.5 mmol, 0.276 ml) in absolute ethanol (5 ml) containing triethylamine (5 mmol, 0.7 ml) to the previous mixture and further irradiated under ultrasonication waves at 50 °C for 20 min.Next, a solution of 6-methyl-3formylchromone (3) (2.5 mmol, 0.47 g) in absolute ethanol (15 ml), was added and heated at 50 °C for 15 min under ultrasonication waves.The mixture was le to cool into room temperature.The formed solid was ltrated off and washed with water.The pure products were obtained aer crystallization from ethanol.In vitro cytotoxicity The American type of culture collection (ATCC) provided human cell lines for human prostate cancer cell line (PC3), liver cancer cells (HepG2), and colon cancer cells (HCT116).A humidied, 5% (v/v) CO 2 atmosphere was used to culture the cells at 37 °C in RPMI-1640 supplemented with (100 mg ml −1 ); penicillin (100 units per ml); and heat-inactivated fetal bovine serum (10% v/v).
Using the sulforhodamine B (SRB) assay, the cytotoxicity of the synthesized compounds against (PC3, HepG2, and HCT116) human tumor cells was assessed.Before being treated with the synthesized compounds, cells growing at 80% conuency, trypsinized and cultured in a 96-well tissue culture plate for 24 h.Cells were subjected to six different doses of each chemical (0.01, 0.1, 1, 10, and 1000 mg ml −1 ), with untreated cells added as a control.Before the cells were xed with TCA (10% w/v) for an hour at 4 °C, they were exposed to the concentrations for 72 h.Aer multiple washings, cells were stained with a 0.4% (w/ v) SRB solution for 10 min in the dark.The surplus stain was eliminated using 1% (v/v) glacial acetic acid.The SRB-stained cells were dissolved in Tris-HCl buffer aer drying overnight.
A microplate reader was used to gauge the color intensity at 540 nm.Sigma Plot 12.0 soware was used to examine the association between each tumor cell line's viability percentage and compound concentrations to determine the IC 50 (drug dose that reduces survival to 50%). 51,52lecular docking The bioactive compounds were subject to docking study to explore their binding mode towards vascular endothelial growth factor kinase insert domain receptor (KDR) (PDB ID: 3EWH) protein which was downloaded from protein data bank.
5][56][57] The docking cavities were dened according to the interactions of protein with the co-crystalized ligands which are also used as reference ligands.The grid box with dimensions of 14 × 16 × 14, with 1.0 Å spacing were placed to make the entire binding cavities involved.The co-crystalized ligands were redocked to the receptor to validate the docking parameters.Docking was performed using AutoDockVina. 58The 2D and 3D images were generated by Discovery Studio and Chimera. 59

Conclusion
We successfully reported to synthesize a novel series of polycyclic systems consisting of carbazole-thiazolidinone-chromone and carbazole-thiazolidinone-pyrazole hybrids as new promising antiproliferative agents.The suggested method was very effective and produced pure products in excellent yields, short times, and in simple laboratory method.Among all the tested products, both products compounds 5b (R = CH 2 -CH] CH 2 ) and 5e (R = 4-ClC 6 H 4 ) which belong to carbazole-thiazolidinone-pyrazole hybrids were found to be the most active against HCT116, PC3 and HepG2 cancer cells.These results were further conrmed by the molecular docking studies, which indicated that the three products have good binding with bind with VEGFR-2 receptor.These bioactive products require further biological studies in the hope that they will be effective and promising anticancer substances.

Fig. 1
Fig. 1 The chemical structures of some carbazole-based thiazole and pyrazole at the C-3 position.

Fig. 2
Fig. 2 Some chemical structures of our published compounds that have chromone and pyrazole compounds with their potent anticancer activities.
pyrazole system 5b showed the OH, NH and C]O thiazolidinone functions at 3256, 3166 and 1695 cm −1 , respectively.The absence of C]O chromone and appearance of OH and NH groups conrmed the ring opening and ring closure.The 1 H-NMR spectrum of 5b displayed four specic singlets at d 13.36, 9.77, 7.83 (d, J = 2.4 Hz) and 7.45 ppm due to OH, NH, H-5 pyrazole and ]CH exocyclic protons, respectively. 49Further, in the 1 H-NMR spectrum of this compound, the allylic protons was observed at d 4.53 (d, J = 5.2 Hz, CH 2 ), 5.25 (dd, J = 6.4 and 1.2 Hz, ]CH 2 ) and 5.95-6.04(m, ]CH).The signals of the methyl and ethyl groups appeared at d 2.24 (NCH 3 ) and 1.36, 4.46 (NCH 2 CH 3 )ppm.Furthermore, the 13 C-NMR spectrum of 5b exhibited signals of C]O thiazolidinone , C-3 pyrazole and C-5 pyrazole , carbon atoms at d 166.4,149.8 and 140.6 ppm, respectively. 50The C-2 thiazolidinone and ]CH exocyclic carbons resonated at d 162.7 and 126.5 ppm, respectively.Besides, the allylic carbons were exhibited at d 45.0 (CH 2 ), 116.4 (]CH 2 ) and 140.3 (]CH).Finally, its molecular ion peak M + was displayed at m/z 533 (31%) in its mass spectrum.The steps and possible mechanisms for the formation of the target products were depicted in Scheme 4. 3-Amino-9ethylcarbazole (1) underwent a nucleophilic addition to a series of isothiocyanates 2a-e in ethanol at room temperature for 15 min under ultrasound irradiation, to form the corresponding carbazolyl-thioureas A which can exist in the tautomeric form B. The thiol group SH attacked the CH 2 group in ethyl bromoacetate to release HBr molecule to form the intermediate C. The later intermediate underwent cyclization through nucleophilic attack of NH group at ester group (intermediate D), followed by removal of ethanol molecule to give the carbazolyl-thiazolidinones E. In the next step, the intermediate E was deprotonated with the help of triethylamine to attack the formyl group of compound 3 (intermediate F), followed by removal of water molecule to isolate the carbazole-thiazolidinone-chromones 4a-e (Scheme 4).The C-2 positions of chromone ring are more electrophilic centers and can be attacked by hydrazine hydrate to undergo ring opening (intermediate H).The ring closure of the latter intermediate H by nucleophilic attack of NH 2 group at the ketonic C]O and releasing of water molecule afforded the nal carbazole-thiazolidinone-pyrazoles 5a-e (Scheme 4).

Fig. 3
Fig.3The chemical structures of the synthetic compounds 4a-e and 5a-e and their yields.

Table 2
The in vitro anticancer activity of products 4a-e and 5a-e against HCT-116, PC3, HepG-2 and 3T3-L1 cancer line cells b IC 50 values are the mean ± SD of three separate experiments.b ND: not determined.